BISC314:Full Protocol
Laboratory Protocols
LAB #1: Learning Sterile Technique and Field Trip to the Cheese Shop
Today we will be taking a trip in to Cambridge to visit Formagio Kitchen, a famous cheese shop in the area - they even have a cheese cave! Each of you will pick a cheese to be your microbial habitat for the next few weeks. We will sample a large variety and learn about where these cheeses came from. Let's be sure we have representative cheeses from these four main classes:
1. A blue cheese
2. A fresh cheese
3. A washed rind cheese
4. A soft, brie-like cheese
Back in the lab, we'll review sterile technique and inoculate media of various kinds from our cheese rinds. We will isolate a beautiful array of different microbes (both eukaryotic and bacterial) from these cheeses and in the next few weeks you'll be using them to investigate two major microbial community functions: growth interactions and chemical signaling.
LAB #2: Macroscopic and Microscopic observation of Isolates
You will have many different colonies growing up on your plates from last week. In your lab notebook, take some time to look at your colonies and describe their morphology, color, and smells! Does your Camembert inoculum smell like Camembert? You may find the following link useful for colony morphology descriptions: ASM MicrobeLibrary
Let's also look at our isolates under the microscope. We will make smears of our organisms next. Before we get to that point, however, it's worth discussing cellular morphology a bit. For the most part, bacteria are much smaller (0.2 to 4 µm) than eukaryotes (~100 µm). We will be using the 100x objectives to see bacterial morphology under the scope. You may be able to see the shapes of many eukaryotes you've isolated under 40x magnification.
Background
The morphological characteristics of bacteria, including size, shape, and arrangement, can be seen by staining a bacterial smear so that individual bacterial cells are distinguished. For most species, morphologic characteristics are genetically determined and, thus, typical of the species. Generally, bacteria range in size from approximately 0.2 µm to 3.0 µm. The basic shapes are spherical (coccus), rod-shaped (bacillus), curved (vibrio), or helical (spirillum, spirochete). However, there are some species of bacteria that show considerable within species variation (termed pleomorphism). For example, both Mycoplasma (bacteria lacking the rigid cell wall of most bacteria) and Arthrobacter (a type of soil bacteria) show forms ranging from coccoid (round) to rodlike to filamentous. Some pathogenic species such as Mycobacterium tuberculosis and Corynebacterium diptheriae are also pleomorphic.
Bacteria are often individual but some species take on a group arrangement based on the way cell division and subsequent separation of the daughter cells occur. Most of the Gram-negative bacilli (rods) are found singly (Escherichia coli) or, sometimes, characteristically in pairs (Klebsiella pneumoniae). The cocci, Streptococcus pneumoniae and Neisseria are called diplococci because they tend to pair. Bacilli found in chains include some Bacillus species. The coccus genera that most often form chains are named Streptococcus for this defining arrangement. A few cocci make regular packets of four or eight (Micrococcus) and some are seen in irregular clumps that resemble bunches of grapes (Staphylococcus). Short rods that form parallel lines, called palisades, include the species that causes diptheria, Corynebacterium diptheriae. Chinese character formation describes a sharply angled bacterial arrangement. Because of their waxy cell walls, Mycobacterium species are difficult to emulsify and tend to stick together in clumps. The pathogen in this group, Mycobacterium tuberculosis, may form long cords of cells.
Keep in mind that individual cells may show deviations from these standard forms. For example, cocci of Neisseria show flattened sides, making them bean-shaped. The rods of Corynebacterium and Mycobacterium often appear club-shaped, with swollen ends or knobs. Both groups may show irregular staining. The diplococci of Streptococcus pneumoniae often appear slightly elongated and lancet-shaped (with one flattened end and one tapered end).
Making a Bacterial Smear
Activity 2: Prepare a Bacterial Smear Slide of Serratia marcescens and Staphylococcus epidermidis and a mixture of both bacteria.
This protocol is found below and at BISC209: Preparing a bacterial smear slide in the Protocols section of this wiki.
Preparing a bacterial smear slide
1. Label a clean, glass slide with a graphite pencil on the far left of the slide with SE, SM, MIX. (The decolorizer in the Gram stain can remove your labels if you use pen or wax pencil.) By convention, labels (top to bottom) match smears (left to right).
2. Place three very small loopfuls of deionized water on the slide as far from each other as possible. (You can use the deionized water bottle on your bench; remove the cover and dip your loop in since sterility is not required for this step.
3. Flame the loop, allow it to cool for a few seconds and touch the cooled loop to a colony of S. epidermidis , picking up a TINY bit of white growth from the bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony is fine.
4. Place the loop with the bacterial growth into the drop of water on far left of the slide. Use a circular motion to make a smooth suspension of the bacteria in the water. Stop when there is a circle of emulsified bacteria about the size of a nickle on the slide. Be sure to leave room for the adjacent drop of water to be spread to a similar size without mixing the two smears.
5. Reflame the loop.
6. Repeat step 4 with the Serratia marcescens in the middle drop of water and then, without flaming your loop, touch the loop to the drop of water on the far right and mix briefly.
7. Reflame your loop and touch it to a Staphylococcus colony again. Place the loop in the far right drop of water mixing it with the Serratia and spread the drop as in step 4 to create a mixed smear.
6. Allow the slide to air dry completely! Be sure all the water on the slide has evaporated before proceeding to heat fixation!!! This drying step is crucially important. If you are impatient, you will "explode" the cells in the next step .
7. Heat fix (to kill and attach organisms to the slide) by quickly passing the slide (smear side up) through a flame 3 times. Use a clothes pin or slide holder and avoid contact with hot glass.
